The long-term objective of this proposal is to elucidate the signaling pathways and cellular mechanisms underlying myogenic phenomena, in particular the role of calcium (Ca2) in the initiation and maintenance of arteriolar constriction following a step change in transmural pressure. The specific aims of this study are to determine: 1) the relationship between smooth muscle Ca2 and arteriolar lumen diameter as a function of the distending transmural pressure, 2) the extent to which Ca2 derived from intracellular stores participates in myogenic activity, and 3) the dependence of the magnitude of Ca2 release from intracellular stores on the size of the step change in transmural pressure and existent level of myogenic tone. For these studies hamster cheek pouch arterioles will be isolated, cannulated, and subjected to step changes in transmural pressure. This in vitro pressurized model is advantageous since the effect of altering the distending pressure on vascular smooth muscle can be determined directly, that is, independent of neural and humoral influences. During select experiments, Ca2 will be depleted from the intracellular stores to assess the relative involvement of Ca2 derived from this source in mediating myogenic behavior. Cytosolic Ca2 concentration ([Ca2+]) will be measured using the Ca2+-sensitive florescent indicator fura-2 AM, with changes in [Ca2+] being assessed by changes in the ratio of emitted fluorescence intensity (R240/380) during alternate excitation at 340 and 380 nm. Simultaneous measurement of arteriolar lumen diameter will be made using a closed-circuit video- microscopy system coupled to a video caliper. The elucidation of these pathways will not only provide an understanding of important vasomotor phenomena, but will also permit an understanding of the potential for myogenic activity of contribute to the changes in vascular resistance that occur in arterial hypertension.